Development of an immunobiotic evaluation system using bovine mammary epithelial cells

Abstract

Mastitis, the inflammation of mammary glands, is one of the most costly diseases of dairy industry, which is often caused by pathogenic bacteria. Antimicrobials are applied for the treatment and prevention of bovine mastitis but their indiscriminate use arouses concerns of residual contamination and increased risk of emerging drug-resistant bacteria. Immunoregulatory probiotics (immunobiotics) are considered as potential substitute for antimicrobials for the prevention of bovine mastitis. In this work, an in vitro system for the evaluation of immunobiotics with beneficial effects against mastitis was developed by using an originally established bovine mammary epithelial cell line (BMECs). BMECs ability to respond to TLR4 activation was evaluated by stimulating cells with LPS for 12h and analyzing the global gene expression changes with a porcine microarray-based transcriptomic assay. A total of 429 genes were differently expressed after LPS stimulation and the most significant change was the up-regulation of inflammatory cytokines and chemokines (p<0.05). The enhanced expressions of IL-1β, IL-8/CXCL8, MIP2-α/CXCL2, MIP2-β/CXCL3, and MCP-1/CCL2 in BMECs after TLR4 activation were confirmed by qRT-PCR. The ability of lactobacilli isolated from feed-lot environment to modulate TLR4-mediated immune response in BMECs was also studied. BMECs were stimulated with Lactobacillus acidophilus CRL2074, L. mucosae CRL2069, L. fermentum CRL2085, or L. rhamnosus CRL2084 for 12h, 24h or 48h and then challenged with LPS for 12h. A strain dependent ability to differentially modulate inflammatory cytokines and chemokines in lactobacilli-treated BMECs was observed. The results indicate that BMECs can be used as a useful in vitro evaluation system for immunobiotics evaluation.